Marijuana is the most widespread illegal drug of abuse in Western societies. Its main active ingredient, delta 9 tetrahydrocannabinol (THC), acts by binding to specific membrane receptors, called cannabinoid receptors. We have learned much in recent years on the pharmacology of these receptors, but we still know very little about their possible pathophysiological roles. Indeed, although a family of endogenous substances that activate cannabinoid receptors (anandamide, 2-arachidonyglycerol, and palmitylethanolamide) have been identified, we don't know whether these substances are released in the brain of freely-moving animals, and what stimuli trigger their release. We propose to use microdialysis and gas chromatography/mass spectrometry techniques to test the hypothesis that endogenous cannabinoid substances are released in vivo by neural activity and by activation of neurotransmitter receptors. The first aim of the proposed research is to determine whether neural activity stimulates the release of distinct endocannabinoid substances in distinct regions of the rat brain. Preliminary experiments have shown that, in dorsal striatum a brain region involved in the control of cognition and movement --, neural activity selectively stimulates the release of anandamide, but not of 2-arachidonylglycerol and palmitylethanolamide. The proposed studies will extend these investigations to other brain areas that have been implicated endogenous cannabinoid functions. The second aim is to determine whether neurotransmitter receptors are linked to endogenous cannabinoid release. Initial results have demonstrated that activation of dopamine D2-like receptors dramatically stimulates anandamide outflow in dorsal striatum. We will use pharmacological approaches to identify the specific dopamine D2-like receptor subtype (D2, D3, and D4) involved in this response, and to define the possible role of D1-like receptors (D1 and D5). The third aim is to examine the effects of cocaine on endocannabinoid release. Cocaine's reinforcing effects may be due to its ability to increase extracellular dopamine, which in turn activates D1-like and D2-like receptors. We will determine the effects of acute and chronic cocaine administration on anandamide release in vivo, and on the levels of anandamide-metabolizing enzymes ex vivo. The forth aim of this proposal is to understand how are endocannabinoid substances inactivated in vivo. Previous in vitro experiments suggest that the biological effects of anandamide may be terminated by uptake into cells followed by enzyme-mediated breakdown. We will use selective uptake and breakdown inhibitors to investigate these processes in vivo. In conclusion, by determining the mechanisms that underlie endogenous cannabinoid release and inactivation in the brain of freely moving animals, our studies will shed new light on the possible pathophysiological roles of the endogenous cannabinoid system, as well as on the mechanism of marijuana abuse. Moreover, by uncovering the neurochemical interactions between cocaine and the endogenous cannabinoids, our studies will contribute to the general understanding of substance abuse and neuropsychiatric disorders.